spillover_compute-flowSet-method: Compute Spillover Matrix - flowSet Method
In DillonHammill/cytoSuite: Compensation, Gating & Visualisation Toolkit for Analysis of Flow Cytometry Data

Description Usage Arguments Details Value Author(s) References See Also Examples

spillover_compute uses the method described by Bagwell & Adams 1993 to calculate the fluorescent spillover matrix using a reference universal unstained control and single stain compensation controls.

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## S4 method for signature 'flowSet'
spillover_compute(x, axes_trans = NULL,
  channel_match = NULL, spillover = "Spillover-Matrix.csv", ...)

`x`	object of class `flowSet` containing pre-gated single stain compensation controls and a universal unstained control. Currently, spillover_compute does not pre-gate samples to obtain a homogeneous cell population for downstream calculations. We therefore recommend pre-gating samples based on FSC and SSC parameters prior to passing them to spillover_compute (i.e. `x` should contain events for single cells only). Passing raw files to spillover_compute will result in inaccurate calculations of fluorescent spillover matrix.
`axes_trans`	object of class `transformList` generated by `estimateLogicle` to transform fluorescent channels for gating. `axes_trans` is required if logicle transformation has already been applied to `x` using estimateLogicle. `spillover_compute` will automatically call `estimateLogicle` internally to transform channels prior to gating, if `axes_trans` is supplied it will be used for the transformation instead.
`channel_match`	name of .csv file containing the names of the samples in a column called "name" and their matching channel in a column called "channel". `spillover_compute` will the guide you through the channel selection process and generate a channel match file called "Compensation-Channels.csv" automatically. If you already have a complete channel_match and would like to bypass the channel selection process, simply pass the name of the channel_match to this argument (e.g. "Compensation-Channels.csv").
`spillover`	name of the output spillover csv file, set to `"Spillover-Matrix.csv"` by default.
`...`	additional arguments passed to `cyto_plot,flowFrame-method`.

Calculate spillover matrix using flowSet containing gated single stain compensation controls and an unstained control. spillover_compute begins by the user selecting which fluorescent channel is associated with each control from a dropdown menu. Following channel selection, spillover_compute runs through each control and plots the density distribution of the unstained control in red and the compensation control in blue. Users can then gate the positive signal for spillover calculation using an interval gate. The percentage spillover is calculated based on the median fluorescent intensities of the stained populations and the universal unstained sample. The computed spillover matrix is returned as an R object and written to a named .csv file for future use.

spillover matrix object and "Spillover Matrix.csv" file.

Dillon Hammill, Dillon.Hammill@anu.edu.au

C. B. Bagwell \& E. G. Adams (1993). Fluorescence spectral overlap compensation for any number of flow cytometry parameters. in: Annals of the New York Academy of Sciences, 677:167-184.

cyto_plot,flowFrame-method

library(CytoRSuiteData)

# Bypass directory check for external files
options("CytoRSuite_wd_check" = FALSE)

# Don't run - skips the gating process
options("CytoRSuite_interact" = FALSE)

# Load in compensation controls
fs <- Compensation
gs <- GatingSet(Compensation)

# Gate using gate_draw
gt <- Compensation_gatingTemplate
gating(gt, gs)

# Channel match fille
cmfile <- system.file("extdata",
  "Compensation-Channels.csv",
  package = "CytoRSuiteData"
)

# Compute fluorescent spillover matrix
spill <- spillover_compute(getData(gs, "Single Cells"),
  channel_match = cmfile,
  spillover = "Example-spillover.csv"
)

# Compensate samples
gs <- compensate(gs, spill)

# Return CytoRSuite_wd_check to default
options("CytoRSuite_wd_check" = TRUE)

# Return CytoRSuite_interact to default
options("CytoRSuite_interact" = TRUE)